Permucous preparation

ABSTRACT

A composition for permucosal administration characterized by containing Antago-3 or a physiologically acceptable salt thereof, and a sucrose fatty acid ester. 
     With the composition for permucosal administration of the invention there is provided a long-term stable preparation having the high permucosal absorption of physiologically active peptide Antago-3 without irritation.

TECHNICAL FIELD

The present invention relates to a composition containing a compoundrepresented by the following formula:

Pmp: β,β-(3-thiapentamethylene)-p-mercaptopropionic acid

D-Trp: D-tryptophan

Pen: L-penicillamine

Ile: L-isoleucine

Asn: L-asparagine

Arg: L-arginine

Gln: L-glutamine

Pro: L-proline

(hereunder referred to as “Antago-3”) or a physiologically acceptablesalt thereof, and more specifically it relates to a permucosalpreparation containing as an effective component Antago-3 or aphysiologically acceptable salt thereof.

BACKGROUND ART

Antago-3 is a synthetic peptide with a molecular weight of 1172.47,which is an oxytocin analog with a powerful and specific oxytocinantagonism effect (WO94/25485-A).

Physiologically active peptides readily undergo enzymolysis by peroralor permucosal administration, while they are virtually unabsorbed bypercutaneous administration due to their low membrane permeability, andhence they are generally administered in the form of injections.

For permucosal administration of physiologically active peptides,expression of their drug effects has required a method whereby an enzymeinhibitor or protective substance is added to protect the drugs fromenzymes, or a method whereby an absorption enhancer is added to promoteabsorption of the drugs before their degradation.

As permucosal preparations of physiologically active peptides there areknown preparations containing cyclodextrin (Japanese Patent PublicationNo. 19092 of 1990), preparations containing bile acid (Japanese PatentPublication No. 25068 of 1994, Japanese Laid-open Patent PublicationsNo. 2932 of 1988 and No. 194260 of 1993), preparations containingsurfactants (Japanese Laid-open Patent Publications No. 130820 of 1984and No. 185030 of 1987), preparations containing absorption accelerators(Japanese Laid-open Patent Publications No. 267528 of 1986 and No.196524 of 1988), preparations containing sucrose fatty acid esters(Japanese Laid-open Patent Publications No. 39822 of 1988, No. 214 of1990 and No. 101020 of 1990), preparations containing saccharides(Japanese Laid-open Patent Publication No. 502920 of 1991) andpreparations containing ethylenediamine tetraacetate (Japanese Laid-openPatent Publication No. 306921 of 1990), but no permucosal preparation isknown that contains Antago-3 or a physiologically acceptable saltthereof as an effective component.

DISCLOSURE OF INVENTION

When an aqueous solution (10 μg/kg) of Antago-3 acetate (hereunderreferred to simply as “Antago-3”) was intravenously administered torats, the response rate on uterine motility was 30% after 30 minutes,and the response rate was 5% after one hour.

However, with pernasal administration of the Antago-3 aqueous solution,it was not possible to express a similar drug effect with intravenousadministration even at a dose of 400 μg/kg.

When polyoxyethylene polyoxypropylene ether, lecithin, sodium caprylate,sodium caprate, aprotinin or bacitracin, sodium ethylenediaminetetraacetate, sodium deoxycholate, β-cyclodextrin, sodium salicylate ordisodium glycyrrhizinate was added to the Antago-3 aqueous solution asan absorption enhancer, it was not possible to express the drug effectobtained by intravenous administration with any of these other thansodium deoxycholate. Incidentally, sodium deoxycholate was shown to havean irritating effect on the nasal cavity mucosa.

The absorption enhancing effect of adding polysorbate 80,polyoxyethylene sorbitan monolaureate, taurine or N,N-dimethylacetamide(hereunder, “DMA”) to the Antago-3 aqueous solution was evaluated basedon the Antago-3 blood concentration and blood concentration area underthe curve (hereunder, “AUC”), but no absorption enhancing effect wasexhibited by any of the compounds.

For permucosal administration of an absorption enhancer-added Antago-3liquid preparation, it is necessary that the presence of the additivecause no non-uniformity such as clouding or precipitation, or irritationat the site of administration.

The present inventors have conducted diligent research on permucosalpreparations containing Antago-3 as the effective component, and as aresult we have found that when a composition containing Antago-3 and asucrose fatty acid ester (hereunder referred to a “composition of theinvention”) is administered, the Antago-3 is very efficiently absorbedthrough the nasal mucosa and rectal mucosa, thus allowing expression ofa drug effect.

However, it was not easy to maintain a transparent state for an aqueouspreparation containing the composition of the invention, due to cloudingor precipitation of the sucrose fatty acid ester.

Upon conducting diligent research on Antago-3-containing aqueouspreparations that maintain transparency, the present inventors thenfound, surprisingly, that the preservative benzalkonium chloride(hereunder, “BZCL”) has a preventive a effect against clouding andprecipitation of the sucrose fatty acid ester while also causingvirtually no mucosal irritation so that it is highly safe for the body,and the present invention has thus been completed.

The composition for permucosal administration provided by the inventioncomprises a sucrose fatty acid ester in a proportion of 0.25-50 parts byweight to one part by weight of Antago-3. The fatty acid of the sucrosefatty acid ester may be stearic acid, palmitic acid, myristic acid,lauric acid, etc., and esters, diesters and triesters of these fattyacids, or mixtures thereof, may be used.

The preparation form may be a solution preparation or other type ofliquid preparation, or a powder preparation.

BZCL may be used as a preservative that can be added to the aqueouspreparation as a solution preparation, and it is combined at 0.03-0.5part by weight, and preferably 0.04-0.16 part by weight of BZCL to onepart by weight of the sucrose fattyacidester.Asisotonizingagentstheremaybementioned polyols such as glycerin andpropylene glycol, saccharides such as mannitol, and nicotinamide, and asviscosity agents there may be mentioned hydroxypropylcellulose(hereunder, “HPC”), polyvinylpyrrolidone (hereunder, “PVP”),carboxymethylcellulose sodium (hereunder, “CMC-Na”) and polyvinylalcohol (hereunder, “PVA”).

A powder preparation may be administered by a method whereby a mixtureof the composition of the invention with lactose, sucrose, mannitol,sorbitol, crystalline cellulose, starch or a low-substituted HPC issprayed by air power, or a method of spraying with an appropriatevolatile carrier.

After adding the preservative, viscosity agent, surfactant, etc. to thecomposition of the invention, it may be filled into a container and usedas a rectal preparation or vaginal preparation. As preservatives theremay be mentioned p-hydroxybenzoic acid ester, BZCL, chlorobutanol andsodium dehydroacetate, as viscosity agents, HPC, PVP, CMC-Na, PVA andcarboxyvinyl polymer, and as surfactants, hydrogenated castor oil andmacrogol.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be described by way of productionexamples. These examples, however, are not intended to be restrictive onthe invention.

COMPARATIVE EXAMPLE 1

Antago-3 (10.0 mg) was dissolved in 100 mL of distilled water to make a0.01% Antago-3 aqueous preparation.

COMPARATIVE EXAMPLES 2-4

According to the method of Comparative Example 1 there were prepared0.4% Antago-3 (Comparative Example 2), 0.2% Antago-3 (ComparativeExample 3) and 0.04% Antago-3 (Comparative Example 4) aqueouspreparations.

COMPARATIVE EXAMPLE 5

A 1 mL aqueous solution of 0.16% Antago-3 and a 1 mL aqueous solution of1.0% polyoxyethylene polyoxypropylene ether were thoroughly mixed tomake a 0.08% Antago-3 aqueous preparation containing 0.5%polyoxyethylene polyoxypropylene ether.

COMPARATIVE EXAMPLES 6-20

According to the method of Comparative Example 5 there were made 0.08%Antago-3 aqueous preparations containing 0.5% lecithin (ComparativeExample 6), 0.5% sodium caprylate (Comparative Example 7), 0.5% sodiumcaprate (Comparative Example 8), 0.1% aprotinin (Comparative Example 9),0.1% bacitracin (Comparative Example 10), 1.0% sodium ethylenediaminetetraacetate (Comparative Example 11) and 1.0% sodium deoxycholate(Comparative Example 12), a 0.04% Antago-3 aqueous preparationcontaining 0.5% sodium deoxycholate (Comparative Example 13), and 0.08%Antago-3 aqueous preparations containing 0.5% β-cyclodextrin(Comparative Example 14), 0.1% sodium salicylate (Comparative Example15), 1.0% disodium glycyrrhizinate (Comparative Example 16), 0.5%polysorbate 80 (Comparative Example 17), 0.5% polyoxyethylene sorbitanmonolaurate (Comparative Example 18), 1.0% taurine (Comparative Example19) and 3.0% DMA (Comparative Example 20).

COMPARATIVE EXAMPLE 21

After thoroughly mixing 1 mL of a 1.0% Antago-3 aqueous solution and 1mL of a 1.0% sucrose fatty acid ester (stearic acid ester) solution, themixture was lyophilized to obtain an Antago-3/sucrose fatty acid ester(1/1) composition for permucosal administration. To this there wereadded to dissolution 1 mL of a 5.0% glycerin aqueous solution and 1 mLof a 1. 0% benzyl alcohol aqueous solution, to make an aqueouspreparation of the composition for permucosal administration.

COMPARATIVE EXAMPLE 22

The Antago-3/sucrose fatty acid ester (1/1) lyophilized composition forpermucosal administration of Comparative Example 21 was dissolved in anaqueous solution prepared by adding distilled water to 1 mL of a 5.0%glycerin aqueous solution, 0.2 mL of a 0.2% methylparaben aqueoussolution and 0.2 mL of a 0.4% propylparaben aqueous solution, for atotal of 2 mL, to make an aqueous preparation of the composition forpermucosal administration.

COMPARATIVE EXAMPLE 23

Distilled water was added to 1 mL of a 5.0% Antago-3 aqueous solutionand 1 mL of a 1.0% sucrose fatty acid ester (stearic acid ester) to avolume of 10 mL, to make an aqueous preparation of an Antago-3/sucrosefatty acid ester (1/0.2) composition for permucosal administration.

COMPARATIVE EXAMPLE 24

Distilled water was added to a mixture of 1 mL of a 0.8% Antago-3aqueous solution, 5 mL of a 1.0% sucrose fatty acid ester (stearic acidester) aqueous solution, 2 mL of a 12.5% glycerin aqueous solution and0.25 mL of a 0.4% BZCL aqueous solution to a volume of 10 mL to make anaqueous preparation of an Antago-3/sucrose fatty acid ester (1/6.25)composition for permucosal administration.

EXAMPLE 1

After thoroughly mixing 1 mL of a 0.16% Antago-3 aqueous solution and 1mL of a 1.0% sucrose fatty acid ester (stearic acid ester) aqueoussolution, the mixture was lyophilized to obtain an Antago-3/sucrosefatty acid ester (1/6.25) composition for permucosal administration.

This was dissolved in 2 mL of distilled water to make an aqueouspreparation of the composition for permucosal administration.

EXAMPLE 2

After thoroughly mixing 1 mL of a 0.08% Antago-3 aqueous solution and 1mL of a 1.0% sucrose fatty acid ester (palmitic acid ester) aqueoussolution and lyophilizing the mixture, the resulting Antago-3/sucrosefatty acid ester (1/12.5) composition for permucosal administration wasdissolved in 2 mL of distilled water to make an aqueous preparation ofthe composition for permucosal administration.

EXAMPLE 3

After thoroughly mixing 1 mL of a 0.04% Antago-3 aqueous solution and 1mL of a 1.0% sucrose fatty acid ester (stearic acid ester and palmiticacid ester at weight ratio of 1:1) aqueous solution and lyophilizing themixture, the resulting Antago-3/sucrose fatty acid ester (1/25)composition for permucosal administration was dissolved in 2 mL ofdistilled water to make an aqueous preparation of the composition forpermucosal administration.

EXAMPLE 4

After thoroughly mixing 1 mL of a 0.04% Antago-3 aqueous solution and 1mL of a 0.2% sucrose fatty acid ester (stearic acid ester) aqueoussolution and lyophilizing the mixture, the resulting Antago-3/sucrosefatty acid ester (1/5) composition for permucosal administration wasdissolved in 2 mL of distilled water to make an aqueous preparation ofthe composition for permucosal administration.

EXAMPLE 5

After thoroughly mixing 1 mL of a 0.02% Antago-3 aqueous solution and 1mL of a 1.0% sucrose fatty acid ester (stearic acid ester) aqueoussolution and lyophilizing the mixture, the resulting Antago-3/sucrosefatty acid ester (1/50) composition for permucosal administration wasdissolved in 2 mL of distilled water to make an aqueous preparation ofthe composition for permucosal administration.

EXAMPLE 6

The same treatment was carried out except that the sucrose fatty acidester (stearic acid ester) of Example 1 was replaced with anothersucrose fatty acid ester (sucrose lauric acid ester), and the resultingAntago-3/sucrose fatty acid ester (1/6.25) composition for permucosaladministration was dissolved in 2 mL of distilled water to make anaqueous preparation of the composition for permucosal administration.

EXAMPLE 7

The Antago-3/sucrose fatty acid ester (1/6.25) composition forpermucosal administration of Example 1 was dissolved in an aqueoussolution prepared by adding distilled water to a mixed solution of 1 mLof a 5.0% nicotinamide solution and 0.2 mL of a 0.2% BZCL solution to avolume of 2 mL, to make an aqueous preparation of the composition forpermucosal administration.

EXAMPLE 8

The Antago-3/sucrose fatty acid ester (1/6.25) composition forpermucosal administration of Example 1 was dissolved in an aqueoussolution prepared by adding distilled water to a mixed solution of 1 mLof a 10.0% sorbitol aqueous solution and 0.2 mL of a 0.2% BZCL solutionto a volume of 2 mL, to make an aqueous preparation of the compositionfor permucosal administration.

EXAMPLE 9

The Antago-3/sucrose fatty acid ester (1/6.25) composition forpermucosal administration of Example 1 was dissolved in an aqueoussolution prepared by adding distilled water to a mixed solution of 1 mLof a 5.0% glycerin aqueous solution and 0.2 mL of a 0.2% BZCL solutionto a volume of 2 mL, to make an aqueous preparation of the compositionfor permucosal administration.

EXAMPLE 10

Distilled water was added to 1 mL of a 0.8% Antago-3 aqueous solution, 1mL of a 5% sucrose fatty acid ester (stearic acid ester) aqueoussolution, 2 mL of a 12.5% glycerin aqueous solution, 1 mL of a 0.1%methylparaben-0.2% propylparabenmixed aqueous solution, 3 mL of a 10%HPC aqueous solution and 1 mL of a 0.2% BZCL aqueous solution to avolume of 10 mL to make an aqueous preparation of an Antago-3/sucrosefatty acid ester (1/6.25) composition for permucosal administration.

EXAMPLE 11

Distilled water was added to 1 mL of a 0.8% Antago-3 aqueous solution, 1mL of a 5% sucrose fatty acid ester (stearic acid ester) aqueoussolution, 2 mL of a 12.5% glycerin aqueous solution, 1 mL of a 5% benzylalcohol aqueous solution, 3 mL of a 10% HPC aqueous solution and 1 mL ofa 0.2% BZCL aqueous solution to a volume of 10 mL to make an aqueouspreparation of a composition for permucosal administration.

EXAMPLE 12

After adding 10 mL of a 1.0% sucrose fatty acid ester (stearic acidester) solution, 9.2 g of 2% Hibis Waco 105 (trade name: Wako Junyaku)as a gel base and a 1 mol/L sodium hydroxide aqueous solution (0.8 g) toAntago-3 (16.0 mg), the total was brought to 20 g with distilled waterand thoroughly mixed to make a gel preparation of an Antago-3/sucrosefatty acid ester (1/6.25) composition for permucosal administration.

EXAMPLE 13

Distilled water was added to 1 mL of a 5.0% Antago-3 aqueous solution, 5mL of a 1.0% sucrose fatty acid ester (stearic acid ester) aqueoussolution, 2 mL of a 12.5% glycerin aqueous solution and 2 mL of a 0.4%BZCL aqueous solution to a volume of 10 mL to make an aqueouspreparation of an Antago-3/sucrose fatty acid ester (1/i) compositionfor permucosal administration.

EXAMPLE 14

Distilled water was added to 1 mL of a 0.8% Antago-3 aqueous solution, 5mL of a 1.0% sucrose fatty acid ester (stearic acid ester) aqueoussolution, 2 mL of a 12.5% glycerin aqueous solution and 2 mL of a 0.4%BZCL aqueous solution to a volume of 10 mL to make an aqueouspreparation of an Antago-3/sucrose fatty acid ester (1/6.25) compositionfor permucosal administration.

EXAMPLE 15

Distilled water was added to 1 mL of a 0.8% Antago-3 aqueous solution, 5mL of a 1.0% sucrose fatty acid ester (stearic acid ester) aqueoussolution, 2 mL of a 12.5% glycerin aqueous solution and 1 mL of a 0.4%BZCL aqueous solution to a volume of 10 mL to make an aqueouspreparation of an Antago-3/sucrose fatty acid ester (1/6.25) compositionfor permucosal administration.

EXAMPLE 16

An aqueous preparation of a composition for permucosal administrationwas made by the same procedure in Example 15 except that the 0.4% BZCLaqueous solution was replaced with a 0.2% BZCL aqueous solution.

EXAMPLE 17

Distilled water was added to 1 mL of a 5.0% Antago-3 aqueous solution, 2mL of a 2.0% sucrose fatty acid ester (stearic acid ester) aqueoussolution, 2 mL of a 12.5% glycerin aqueous solution and 1 mL of a 0.2%BZCL aqueous solution to a volume of 10 mL to make an aqueouspreparation of an Antago-3/sucrose fatty acid ester (1/0.8) compositionfor permucosal administration.

EXAMPLE 18 Preparation 1 for Monkey

Distilled water was added to 50 mg of Antago-3, 4 mL of a 2.5% sucrosefatty acid ester (stearic acid ester) aqueous solution, 2 mL of a 0.4%BZCL aqueous solution and 2 mL of a 12.5% glycerin aqueous solution to avolume of 20 mL to make an aqueous preparation of anAntago-3/sucrosefatty acid ester (1/2) composition for permucosal administration.

EXAMPLE 19 Preparation 2 for Monkey

An aqueous preparation of an Antago-3/sucrose fatty acid ester (1/1)composition for permucosal administration was made by the same procedurein Example 18 except that 2 mL of the 2.5% sucrose fatty acid ester(stearic acid ester) aqueous solution was used.

EXAMPLE 20 Preparation 3 for Monkey

An aqueous preparation of an Antago-3/sucrose fatty acid ester (1/0.4)composition for permucosal administration was made by the same procedurein Example 18 except that 0.8 mL of the 2.5% sucrose fatty acid ester(stearic acid ester) aqueous solution was used.

EXAMPLE 21 Preparation 4 for Monkey

An aqueous preparation of an Antago-3/sucrose fatty acid ester (1/0.5)composition for permucosal administration was made by the same procedurein Example 18 except that 100 mg of Antago-3 was used and 2 mL of the2.5% sucrose fatty acid ester (stearic acid ester) aqueous solution wasused.

EXAMPLE 22 Preparation 5 for Monkey

An aqueous preparation of an Antago-3/sucrose fatty acid ester (1/0.25)composition for permucosal administration was made by the same procedurein Example 18 except that 200 mg of Antago-3 was used and 2 mL of the2.5% sucrose fatty acid ester (stearic acid ester) aqueous solution wasused.

EXAMPLE 23

A 10 mg portion of an Antago-3/sucrose fatty acid ester (1/1)lyophilized composition for permucosal administration produced accordingto Comparative Example 21 was thoroughly mixed with 290 mg of lactose. Ahard capsule (#4) was filled with 30 mg of the mixture to make a powderpreparation of the composition for permucosal administration.

EXAMPLE 24

A powder preparation of a composition for permucosal administration wasmade according to the same procedure as Example 23, except that thelactose was replaced with crystalline cellulose.

EXAMPLE 25

After adding 10 mL of a 1.0% sucrose fatty acid ester(palmiticacidester) solution, 9.2 g of 2% Hibis Waco105 (trade name:Wako Junyaku) as a gel base and a 1 mol/L sodium hydroxide aqueoussolution (0.8 g) to Antago-3 (16.0 mg), the total was brought to 20 gwith distilled water and thoroughly mixed to make a gel preparation ofan Antago-3/sucrose fatty acid ester (1/6.25) composition for permucosaladministration.

EXAMPLE 26

A gel preparation of an Antago-3/sucrose fatty acid ester (1/6.25)composition was made by the same procedure as Example 25, except thatthe sucrose fatty acid ester (palmitic acid ester) was replaced withanother sucrose fatty acid ester (myristic acid ester).

[Uterine Motility Response Rate]

(Method)

Estrus was induced in female rats (8 weeks old) with estradiol prior tothe experiment. With each rat under urethane anesthesia, a catheter wasinserted into the uterine horn, and after injection of physiologicalsaline it was connected to a low-pressure transducer. A distortionmeasurement amplifier was used to record a rectigraph of uterinemotility. After continuous intravenous infusion of oxytocin (50mU/kg/min) through the femoral vein and confirmation of a stabilizedcontraction waveform, the test sample was pernasally administered (100μL/kg). The suppressing effect on uterine motility by Antago-3 wasexpressed by the response rate, where 100% was defined as the multiplierof the average contraction frequency and amplitude in 10 minutes for thecontraction waveform with oxytocin alone prior to administration of thesample. A response rate on uterine motility closer to 0% indicates thata stronger effect of Antago-3 was exhibited.

(Results)

The response rates on uterine motility with pernasal administration ofAntago-3 aqueous solutions and aqueous solutions of the compositions ofthe invention are shown in Table 1, with intravenous administration ofAntago-3 used as a control.

TABLE 1 Response rates on uterine motility with pernasal administrationResponse rate (%) Dosage Time (hr) Sample (μg/kg) 0.5 1.0 1.5 2.0Comparative  1  10  30  5 — — (intravenous Example injection)  2 400  80 25  20  20  3 200  90  70  60  50  4  40 100 100 100 100  5  80 100 100100 100  6  80 100 100 — 100  7  80 100 100 — 100  8  80  90  50 —  40 9  80 100 100 100 100 10  80 100 100 100 100 11  80 100 100 100 100 12 80  0  0  0  10 13  40  30  20  15  10 14  80 100 100  80  40 15  80 80  80  80  80 16  80 100  90  80  75 Example  1  80  0  0  0  0  2  40 5  0  0  0  3  20  10  5  5  5  4  20  30  10  10  10  5  10  30  15 10  5

With intravenous administration of the Antago-3 aqueous solution,uterine motility was inhibited with a dosage of 10 μg/kg. With pernasaladministration of the Antago-3 aqueous solution, a dosage of at least400 μg/kg was required to obtain the same drug effect as the intravenousadministration.

The sucrose fatty acid esters had a notable effect of enhanced Antago-3absorption, with the aqueous solutions of the composition of theinvention exhibiting activity roughly equivalent to that observed withintravenous administration.

No absorption enhancing effect was exhibited and uterine motility couldnot be inhibited when polyoxyethylene polyoxypropylene ether, lecithin,sodium caprylate, sodium caprate, aprotinin, bacitracin, sodiumethylenediamine tetraacetate, β-cyclodextrin, sodium salicylate ordisodium glycyrrhizinate was added to Antago-3 as an absorptionenhancer. When sodium deoxycholate was added, an absorption enhancingeffect equivalent to that of the composition of the invention wasexhibited.

[Absorption Enhancement by Blood Concentration AUC Method]

(Method)

(1) Pernasal Administration

A 100 μL/kg dose of the test sample was administered into both nasalcavities of awake 9-week-old female rats on a 16-hour fast, and 0.2 mLof blood was taken from the cervical vein at 15 minutes, 30 minutes, 1hour, 2 hours and 4 hours after administration. The blood wastransferred to an opaque blood collecting tube, and an inhibitor (asolution prepared by dissolving 100, 000 KIU of aprotinin (product ofWako Junyaku) and 4 mg of bestatin (product of Sigma Co.) in 5 mL ofdistilled water at the time of use) in an amount of 1/20 the amount ofblood was silently added along the wall of the collecting tube, afterwhich the blood was held in ice water and within 1.5 hours wascentrifuged (4° C., 1600×g, 15 minutes) to obtain the blood plasma whichwas stored at −80° C. until measurement. The Antago-3 in the plasma wasmeasured by radioimmunoassay (RIA)

(2) Perrectal Administration

The anus was held open with tweezers and the test sample was injected at100 μL/kg in solution form or 100 μg/kg in gel form using an injectiontube or a pipette, after which the anus was closed off with an adhesiveto prevent leakage of the test sample. Blood was taken and measured inthe same manner as for the pernasal administration.

The blood concentration area under the curve (AUC) was determined by thetrapezoid method, and the absorption enhancement was expressed as theratio to the AUC for the sample of <Comparative Example 2> in which noabsorption enhancer was added. The test sample wherein 80 μg/kg ofAntago-3 was administered was evaluated based on the value of 5 timesthe calculated AUC value for comparison with Comparative Example 2 (400μg/kg administration).

(Results)

The results are shown in Tables 2 and 3.

TABLE 2 Blood concentration, AUC and absorption enhancement (pernasaladministration) Absorp- Blood concentration (ng/mL) tion Time (hr)AUC_(o→4 hr) enhance- Sample 0.25 0.5 1.0 2.0 4.0 (ng.hr/mL) mentCompar-  2  3  2  2 2 1  7  1 ative 17  1  0  0 0 0 — — Example 18  7  3 0 0 0  3  2 19  1  0  0 0 0 — — 20  1  0  0 0 0 — — Example  1 60 30 155 1 46 33  6 55 25 15 3 1 38 27  7 65 35 17 5 1 51 36  8 55 25 15 3 1 3827  9 60 35 15 3 1 44 31 10 50 20 10 3 1 31 22 11 55 30 15 5 1 45 32

TABLE 3 Blood concentration, AUC and absorption enhancement (perrectaladministration) Absorp- Blood concentration (ng/mL) tion Time (hr)AUC_(o→4 hr) enhance- Sample 0.25 0.5 1.0 2.0 4.0 (ng.hr/mL) mentCompar-  2 22 15  9  5 2 27 1 ative Example  1 35 27 15 10 1 46 9Example 12 42 27 13  5 1 39 7

The absorption enhancements of the aqueous preparations and gelpreparations of compositions according to the invention were 22-fold to36-fold with pernasal administration and 7-fold to 9-fold withperrectaladministration, compared to those with no addition of an absorptionenhancer.

[Stability of Solution Preparations]

(Method)

Each of the solution preparations (Examples 13-17) made from acomposition of the invention was placed in a transparent stoppered glasstest tube, and stored at 5° C. Appearance were evaluated by visuallyobserving the solution state at 1000 lux under a white fluorescent lamp.

(Results)

The preparations in which benzyl alcohol (Comparative Example 21) andmethylparaben/propylparaben (Comparative Example 22) were added aspreservatives, the preparation in which no preservative was added(Comparative Example 23) and the preparation with low addition of BZCL(Comparative Example 24) exhibited clouding after 3 days of storage, dueto precipitation of the sucrose fatty acid esters. However, thepreparations of Examples 13-17, in which at least 0.04 part by weight ofBZCL was added to one part by weight of the sucrose fatty acid ester,maintained a colorless, transparent state.

[Nasal Mucosa Irritation]

(Method)

Eight-week-old female rats were used, with five per group. The rats wereadministered 100 μL/kg of the test sample into the nasal cavities once aday, for 7 continuous days. On the 8th day the nasal cavities wereextracted, and after dividing them into 3 parts from the nasal cavityopening to the deep end of the nasal cavity, they were fixed withformalin to prepare specimens. After 10-14 days of fixing, ahistopathological examination was made of the state of the mucosa,exudation and any bleeding.

Mild irritation was judged when abnormalities were found in the nasalmucosa at sections near the nasal cavity opening, and moderate to severeirritation was judged when the sections where abnormalities were foundwere further to the deep end. Moderate or greater irritation was alsojudged when degeneration, ablation, exudation or bleeding was found onthe nasal mucosa epithelium at sections near the nasal cavity opening.

As controls there were used physiological saline and commerciallyavailable desmopressin (Kyowa Hakko Kogyo, KK.)

(Results)

The results are shown in Table 4.

TABLE 4 Nasal cavity mucosa irritation Normal Mild Moderate SeverePhysiological 3 2 0 0 saline Desmopressin 5 0 0 0 Comparative 13 1 2 2 0Example 21 4 1 0 0 Example  1 5 0 0 0  2 5 0 0 0  4 5 0 0 0 15 4 1 0 0

In the [Comparative Example 13] group there were observed exudation inthe nasal mucosa and beaker cell proliferation in the respiratoryepithelium, and therefore moderate irritation was determined.

Beaker cell proliferation was found in the respiratory epithelium of onerat in each of the [Comparative Example 21] and [Example 15] groups, butit was milder than in the [physiological saline] group used as acontrol.

No irritation was found in the [Example 1] group, [Example 2] group,[Example 4] group or [desmopressin] group.

No dead animals were found in any of the groups, nor were any abnormalsymptoms found.

[Monkey Absorption Test]

A female monkey with a body weight of 3 kg was strapped to a monkeychair without anesthesia and in a state of rest, and the head was heldso that the external naris was directed straight downward. A fixed-doseatomizing spray apparatus (spray volume: 50 μL/puff) filled with thetest solution was then held vertically upward, and after stabilizing thespray condition by test spraying 4 or 5 times, the tip (about 1 cm) wasinserted into the right nasal cavity and sprayed once for 50 μL. Afterspraying, the external naris was pointed forward and the external nariswhich had received the agent was closed with a finger and held for about10 seconds in order to prevent run-off of the administered testsolution, after which the left nasal cavity was sprayed in the samemanner. The animal had been fasting from the evening of the day prior toadministration, and it was fed after completion of the blood samplingduring the 24 hours after administration.

For blood sampling, 0.3mL of blood was taken from the femoral vein oranterior humeroradial lateral cutaneous vein at 5, 15 and 30 minutes,and 1 and 2 hours (total of 5 times) after administration forpreparations 1, 3, 4 and 5, and at 5, 15 and 30 minutes, and 1, 2, 4, 8and 24 hours (total of 8 times) after administration for preparation 2.

The sampled blood was processed in the same manner as for the pernasaladministration to obtain blood plasma which was stored at −80° C. untilmeasurement.

[Measurement of Blood Concentration]

In an Isolute C18 cartridge (manufactured by Uniflex Co.) there wereadded 0.2 mL-0.05 mL of blood plasma, 0.1 mL of an internal standardsolution (70% methanol solution containing 0.04 ppm reserpine) and 0.5mL of a 0.1 M phosphate buffer solution (pH 7.0) to adsorb thecontaminants in the plasma, and the Antago-3 was eluted out with 2 mL of1-acetic acid-containing methanol. The eluate was distilled underreduced pressure, and 0.2 mL of a methanol:1.0% acetic acid mixture(7:3) was added to and dissolved in the residue to prepare a testsolution. Separately, a bulk agent for quantitation was treated in thesame manner and used as a standard solution. The test solution and thestandard solution were subjected to liquid chromatography to determinethe blood concentration.

TABLE 5 Blood concentrations Blood concentration (ng/mL) 5 min 15 min 30min 1 hour 2 hours Example 18   112   372    346    456    368  ±23 ±105 ±80  ±52  ±78 Example 19   286   491    419    313    407 ±111 ±193 ±105  ±66  ±107 Example 20    74   102    160    96    130  ±23 ±105 ±80  ±52  ±78 Example 21   385 1161   2087   2252   2424 ±152 ±245 ±651  ±592  ±692 Example 22   828 2118   4201   2903   2851 ±345 ±992±2039 ±1128 ±1446 Note: mean value ±S.E. (n = 4)

The aqueous preparations of compositions according to the inventionexhibited average blood concentrations that increased as the sucrosefatty acid ester concentration was higher, from 0.1% (Example 20) to0.25% (Example 19) to 0.5% (Example 18), with a constant bulk agentconcentration, and therefore the absorption enhancing effect depended onthe concentration of the sucrose fatty acid ester. For Example 19, thebioavailability was 33% as determined by the AUC obtained from the bloodconcentrations for 24 hours after administration.

The blood concentrations increased as the bulk agent concentration washigher, from 0.25% (Example 19) to 0.5% (Example 21) to 1.0% (Example22), with a constant sucrose fatty acid ester concentration.

Industrial Applicability

As explained above, a composition for permucosal administrationaccording to the present invention is characterized by containing thephysiologically active peptide Antago-3 or a physiologically acceptablesalt thereof, and a sucrose fatty acid ester, and it has the highpermucosal absorption of Antago-3 without irritation, rendering ituseful as a long-term stable preparation.

What is claimed is:
 1. A permucosal preparation comprising compound I:

Pmp: β,β-(3-thiapentamethylene)-β-mercaptopropionic acid, D-Trp:D-tryptophan Pen: L-penicillamine Ile: L-isoleucine Asn: L-asparagineArg: L-arginine Gln: L-glutamine Pro: L-proline or a physiologicallyacceptable salt thereof, a sucrose C₁₂-C₁₈ fatty acid ester, andbenzalkonium chloride present in a proportion of 0.03 to 0.5 part byweight to one part by weight of the sucrose C₁₂-C₁₈ fatty acid ester. 2.A permucosal preparation according to claim 1, wherein the sucroseC₁₂-C₁₈ fatty acid ester is present in a proportion of 0.25-50 parts byweight to one part by weight of compound I or its physiologicallyacceptable salt.
 3. A preparation according to claim 1 wherein thesucrose C₁₂-C₁₈ fatty acid ester is selected from the group consistingof a sucrose stearic acid ester, sucrose palmitic acid ester, sucrosemyristic acid ester and sucrose lauric acid ester.
 4. A preparationaccording to claim 1, which is suitable for per-nasal mucosaadministration.
 5. A permucosal preparation comprising compound I;

Pmp: β,β-(3-thiapentamethylene)-β-mercaptopropionic acid D-Trp:D-tryptophan Pen: L-penicillamine Ile; L-isoleucine Asn: L-asparagineArg: L-arginine Gln; L-glutamine Pro: L-proline or a physiologicallyacceptable salt thereof, a sucrose C₁₂-C₁₈ fatty acid ester, andbenzalkonium chloride present in a proportion of 0.03 to 0.5 part byweight to one part by weight of the sucrose C₁₂-C₁₈ fatty acid ester,wherein the sucrose C₁₂-C₁₈ fatty acid ester is a mixture of one or moreof sucrose stearic acid ester, sucrose palmitic acid ester, sucrosemyristic acid ester or sucrose lauric acid ester.